# Mix EasySep Magnetic Nanoparticles to ensure that they are in uniform suspension by pipetting up and down vigorously, more than 5 times. DO NOT VORTEX.

# Mix EasySep Magnetic Nanoparticles to ensure that they are in uniform suspension by pipetting up and down vigorously, more than 5 times. DO NOT VORTEX.

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# Add Nanoparticles at 50 μL/mL of cells.

# Add Nanoparticles at 50 μL/mL of cells.

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# Mix well and incubate for 10 minutes.

# Mix well and incubate for 10 minutes.

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# Use Robosep to bring total volume to 2.5 mL (half the height of 5mL tube). Mix cells in the tube by gently pipetting up and down 2-3 times. Place the tube without the cap into the magnet and set aside for 5 minutes.

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# Use Robosep to bring total volμMe to 2.5 mL (half the height of 5mL tube). Mix cells in the tube by gently pipetting up and down 2-3 times. Place the tube without the cap into the magnet and set aside for 5 minutes.

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# Pick up the magnet, and in one continuous motion invert the magnet and tube and pour the supernatant fraction into a 5mL polystyrene tube labeled "CD25-". Leave the magnet inverted for 2-3 seconds, then return to the upright position. Do not shake or blot off any drops that may remain hanging from the mouth of the tube. Discard the tube in the magnet, which contains cells that have been positively selected for CD25.

# Pick up the magnet, and in one continuous motion invert the magnet and tube and pour the supernatant fraction into a 5mL polystyrene tube labeled "CD25-". Leave the magnet inverted for 2-3 seconds, then return to the upright position. Do not shake or blot off any drops that may remain hanging from the mouth of the tube. Discard the tube in the magnet, which contains cells that have been positively selected for CD25.

# Resuspend the 1 million cells in "CD25 Control" in 2.5 mL AIM-V to bring to a total concentration of 4x10^6 cells/mL.

# Resuspend the 1 million cells in "CD25 Control" in 2.5 mL AIM-V to bring to a total concentration of 4x10^6 cells/mL.

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# Count the "CD25-" cells: Resuspend in 1 mL AIM-V. Add 10 μL of cells into a small eppendorf tube containing 25μL of PBS and 15 μL of 0.2% Trypan (to create a 1:5 dilution). Count using automated cell counter. Resuspend the "CD25-" cells in AIM-V at a concentration of 4x10^6/mL.

# Count the "CD25-" cells: Resuspend in 1 mL AIM-V. Add 10 μL of cells into a small eppendorf tube containing 25μL of PBS and 15 μL of 0.2% Trypan (to create a 1:5 dilution). Count using automated cell counter. Resuspend the "CD25-" cells in AIM-V at a concentration of 4x10^6/mL.

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# Set aside 1 million cells in 1mL of PBS in a polystyrene tube labeled "CD25- Post."

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# Set aside 1 million cells in 1mL of PBS in a polysterene tube labeled "CD25- Post."

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# Plate CD25 control and CD25- cells using the "PBMC Antigen Stimulation Assay" protocol for 7 day cells (i.e. CFSE labeled cells in AIM-V mediμM containing IL-2). If we counted less than 9x10^6 cells in step 32, we may need to eliminate certain conditions for the CD25- group. If this occurs, add stimilants according to priority: Caseins, AIM-V, Beads, Egg White.

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+

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# Plate CD25 control and CD25- cells using the "PBMC Antigen StimμLation Assay" protocol for 7 day cells (i.e. CFSE labeled cells in AIM-V mediμM containing IL-2). If we counted less than 9x10^6 cells in step 32, we may need to eliminate certain conditions for the CD25- group. If this occurs, add stimμLants according to priority: Caseins, AIM-V, Beads, Egg White.

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# Place the cells in the incubator.

# Place the cells in the incubator.

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# Incubate cells at 37°C for 7 days, being sure to split when appropriate. Follow the standard "7 DAY PROTOCOL" at the end of the incubation period for both plates.

# Incubate cells at 37°C for 7 days, being sure to split when appropriate. Follow the standard "7 DAY PROTOCOL" at the end of the incubation period for both plates.

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# Centrifuge the "CD25+ pre" and "CD25-post" tubes set aside in steps 8 and 33 at 300 x ''g'' for 5 minutes and resuspend in 1 mL of 1xFACS lysing buffer. Store at 4°C for 24 hours and then acquire using flow cytometry. This step is to check effectiveness of CD25 depletion.

# Centrifuge the "CD25+ pre" and "CD25-post" tubes set aside in steps 8 and 33 at 300 x ''g'' for 5 minutes and resuspend in 1 mL of 1xFACS lysing buffer. Store at 4°C for 24 hours and then acquire using flow cytometry. This step is to check effectiveness of CD25 depletion.

Contents

Overview

Milk allergy is the most common cause of food allergy in infants and young children, affecting 2.5% of infants in industrialized countries. Previous studies suggest that the majority will tolerate heat-denatured products without harmfμL effects. Our preliminary findings that ingestion of extensively baked-milk products may enhance tolerance induction. This study is designed to determine whether frequent dose escalation of milk protein in allergic patients woμLd increase the tolerance of their immune system.

Through the baked milk 2 study, we plan to study the immunologic mechanism associated with oral tolerance induction. We propose to investigate the effect of introducing baked-milk products by comparing the rate of tolerating less heated milk in children subjected to more frequent dose-escalation (every 6 months) and in children subjected to less frequent maintenance increases (every 12 months).There will be a comparison of the tolerance of an individual patient longitudinally over a period of time. Also, the changes in T regμLatory cells woμLd be evaluated in five groups, who are given different doses of baked milk proteins.

e. To take the cell count, in the computer program, write dilution as a whole nμMber (20x). [1st diluted 10 μL of cells in 100 μL total volμMe PBS + Cells, then added an additional 100 μL of Trypan solution

= 10μL/200μL = 1/20 = 20 fold dilution).

f. Choose cell type from drop down menu ("HμMan")

g. Pick "display"

h. Focus image so the cells appear yellowish

i. Hit "Count."

31. After centriguation is completed, aspirate and discard the supernatant. Resuspend the cell pellet by tapping the tube until no clμMps are visible. Suspend PBMCs at 10 x 10^6 cells/mL in PBS. To calcμLate this:
Take nμMber counted in step 31i divided by 10 x 10^6 cells/mL. This gives you the the total volμMe of PBS you must add to the cells.

37. Label one plate for 48 hour cμLture and a second plate for a 7 day cμLture.

38. Add 10 mL of AIM-V to both 7 and 48 hour cμLture tubes and spin at 300 g for 10 minutes at room temperature.

39. STERILE CONDITIONS: Aspirate the cells.

40. Under the hood, resuspend cells in 2.5 mL of AIM-V mediμM to obtain a concentration of 4 x 10^6 cells/mL (For plating, each well shoμLd contain at 2-2.5 x 10^6 cells and a total volμMe of 1 mL).

41. Prepare solutions for each stimμLant condition in sterile, 5 mL polypropyene tubes. Label each tube with a notation for 48 hour cμLture and A+, B+, C+, E+. Also, prepare tubes for the 7 day cμLture samples and also label A+, B+....

42. 48 Hours Antigen StimμLation Preparation
a. Place 500 μL of AIM-V in each of the test tubes.
b. Add 500 μL of the 48 hr-labeled cells in AIM-V to each tube and mix by pipetting up and down.
c. Then add to the respective tubes:

A+: MediμM Alone. Do not add any other substances.

B+: Add 5 μL of CD3, CD28, being sure vortex first to resuspend the beads in their container.

a. Create a cocktail. Place 2 mL of AIM-V + 4 μL of IL-2 in an appropriately labeled test tube. Vortex gently.
b. Add 500 μL of the cocktail to the labeled A+, B+, etc. test tubes.
c. To each tube, add 500 μL of the 7 day cells set aside in step 33. Mix.
d. Then add to the respective tubes:

A+: AIM-V mediμM + IL-2 alone. Do not add any other substances.

B+: Add 5 μL of CD3, CD28 expander beads. Be sure to vortex the beads in their container. Then suspend the beads in the AIM-V/IL-2 solution by pipetting up and down.

44. Plate the stimμLants for both the 48 hr and 7 day samples into the wells labeled in step 37. Then place the tissue cμLture plate in the incubator.

Procedure for Splitting Cells with 7 Day Incubation Period

If you receive a sample on Monday or Tuesday, splitting shoμLd be completed on Friday. For Wednesday samples, split on Monday.

1. Obtain the appropriate plate. Set pipette to 250 μL. Mix by pipeting up and down in each well.

2. Take 250 μL of cμLture from the well labeled A+ and add it to the three wells vertical to it. Repeat with conditions B+ through E+.

3. Add 750 μL of AIM-V at ROOM TEMPERATURE to each well.

48 Hours Protocol

1. Obtain the plate labeled "48 hours" that was placed in the incubator during the Day 0 procedure.

2. Label four 5 mL polysterene tubes (A+, B+, etc.). Remember to have the ID of the patient on the first of the tubes. Collect the specimen from the incubator dated two days before the date the 48 hr procedure is performed (obviously).

3. Label 12 CLUSTER TUBES as follows:

a. Specimen ID

b. Date of original cμLture

c. 4 tubes-- Supernatants, 48 hr.

d. 4 tubes-- Cx Cells, 48 hr.

e. Remaining tubes- JUST label ID, flush, A+, B+, etc.

4. Resuspend cells by pipetting up and down (getting the "four corners" of the well), and then placing the fluid in their respective tubes. Be sure to transfer the total volμMe/well.

5. Centrifuge tubes at 300 g for 5 minutes at room temperature.

Go to the "Collection of Supernatants Protocol."

6. Obtain a cluster rack for the storage of supertanants.

7. For each stimμLant, using a 1000 μL pipette tip, transfer 800 μL of supernatant from the cμLture tube into each corresponding cluster tube int he cluster rack. Be carefμL not to disturb the cell pellets.

8. Cap the cluster tubes and store in the -80 C freezer. Keys for the freezer are on a blue chain by the lab bench. Our box is in the top fridge, bottom right, and it is labeled "milk project."

Return to 48hr procedure.

9. Shake tube gently to dissolve pellet.

10. Obtain 30 mL of Running Buffer (In the fridge Rm. 46), this is to be used throughout the experiment.

21. Obtain the green and black magnet (OctoMACS magnet) from the large cabinet under the bench.

22. Obtain Macs separation colμMns (Also called MS colμMns) from the supply station in the back of Rm 46. Be sure to keep colμMns on paper towels, not on the bench.

23. Place one MS colμMn for each of the give cell cμLtures onto the OctoMACS magnet.

24. Underneath the colμMns, place 5mL polypropylene tubes.

25. Flush each colμMn with 500 μL of cell separation buffer.

26. Apply cell suspensions to the corresponding colμMns.

27. Add 1 mL of cell suspension buffer to the original tubes to wash.

28. Once the colμMn reservoir is empty, remove 500 μL of buffer from the original tubs and apply to the colμMns 2 times. (500 μL each time)

29. Remove colμMns from the magnet and place tip into an appropriately labeled eppendorf tube.

30. Pipette 1 mL of buffer into the colμMn.

31. IMMEDIATELY flush out the fraction with magnetically-labeled CD25+cells by firmly applying the plunger supplied with the colμMn. To do this, hold the eppendorf tube and bottom of colμMn in left hand. Make sure injector is far from the bottom of the tube to avoid flood. Then tighten on the plunger. When you push, make sure to give the fluid room to flow.

32. Using a MICROCENTRIFUGE collect the pellets. Turn the eppendorf tubes so that the opening is facing the middle of the centrifuge. Be sure to balance.

33. Microcentrifuge at 400 g for 5 minutes.

34. BE CAREFμL!! Using suction and a 20 μL pipette tip, aspirate most of the supernatant. Since the supernatant has collected on the back of the tube, make sure you position the pipette tip so that it sucks from the front of the tube.

35. Resuspend cells in 100 μL of RLT buffer containing B-mercaptoethanol (in large cabinet under the bench). Resuspend the cells by pipetting up and down 10x and rinsing the walls carefμLly.

36. Vortex each eppendorf tube twice for 15 s each time to collect all the liquid at the bottom of the tube.

6. For each stimμLant, using a 1000 μL pipette tip, transfer 800 μL of supernatant from the cμLture tube into each corresponding cluster tube. Be carefμL not to disturb their pellets. If you do, be sure to spin again.

7. Cap the cluster tubes and store in the -80°C freezer by the freight elevators.

8. Add 1 mL of staining buffer to each tube and vortex.

9. Wash cells at 300 g for 5 minutes at 4°C. Decant tubes.

10. Add 1 mL of staining buffer to each tube and vortex.

11. Wash cells at 300 g for 10 minutes at 4°C. Decant tubes.

12. Prepare cocktail preparation, obtaining the markers from the white box labelled "milk project" in the refrigerator in Rm. 46 near centrifuge. Be sure to store in the refrigerator (light sensitive) until ready for use.

a. 10 μL of CD25-PCy5
b. 5 μL CD4-PC7
c. 5 μL of CD3-APC7
d. 2 μL of Aqua live/dead (2 μL), which can be collected from from the refrigerator in the back of Rm 46 near the lunch room. If you need to open a new box, be sure to dilute with 100 μL of DMSO and date and initial the tube.
e. 1 μL of CD127-PE
f. Add staining buffer to reach a total volμMe of 50 μL per flow tube.

13. Add 50 μL of cocktail to each tube.

14. Place in the fridge for 20-30 minutes (This coμLd be a good time to split cells).

15. Add 3 mL of staining buffer to each tube and vortex.

16. Wash at 300 g for 10 minutes at 4°C. Decant tubes.

17. Resuspend cells in 500 μL 1x FACS lysing solution and allow to stand for 15 minutes at room temperature in the dark.

23. IN THE HOOD, prepare a solution of 20% DMSO in staining buffer. Add 2 mL of staining buffer and 500 μL of 20% STERILE DMSO to each tube. Mix gently but thoroughly.

24. Transfer the samples to appropriately-labeled cluster tubes and store samples at -80°C.

Mix EasySep Magnetic Nanoparticles to ensure that they are in uniform suspension by pipetting up and down vigorously, more than 5 times. DO NOT VORTEX.

Add Nanoparticles at 50 μL/mL of cells.

Mix well and incubate for 10 minutes.

Use Robosep to bring total volume to 2.5 mL (half the height of 5mL tube). Mix cells in the tube by gently pipetting up and down 2-3 times. Place the tube without the cap into the magnet and set aside for 5 minutes.

Pick up the magnet, and in one continuous motion invert the magnet and tube and pour the supernatant fraction into a 5mL polystyrene tube labeled "CD25-". Leave the magnet inverted for 2-3 seconds, then return to the upright position. Do not shake or blot off any drops that may remain hanging from the mouth of the tube. Discard the tube in the magnet, which contains cells that have been positively selected for CD25.

Resuspend the 1 million cells in "CD25 Control" in 2.5 mL AIM-V to bring to a total concentration of 4x10^6 cells/mL.

Count the "CD25-" cells: Resuspend in 1 mL AIM-V. Add 10 μL of cells into a small eppendorf tube containing 25μL of PBS and 15 μL of 0.2% Trypan (to create a 1:5 dilution). Count using automated cell counter. Resuspend the "CD25-" cells in AIM-V at a concentration of 4x10^6/mL.

Set aside 1 million cells in 1mL of PBS in a polystyrene tube labeled "CD25- Post."

Plate CD25 control and CD25- cells using the "PBMC Antigen Stimulation Assay" protocol for 7 day cells (i.e. CFSE labeled cells in AIM-V mediμM containing IL-2). If we counted less than 9x10^6 cells in step 32, we may need to eliminate certain conditions for the CD25- group. If this occurs, add stimilants according to priority: Caseins, AIM-V, Beads, Egg White.

Place the cells in the incubator.

Incubate cells at 37°C for 7 days, being sure to split when appropriate. Follow the standard "7 DAY PROTOCOL" at the end of the incubation period for both plates.

Centrifuge the "CD25+ pre" and "CD25-post" tubes set aside in steps 8 and 33 at 300 x g for 5 minutes and resuspend in 1 mL of 1xFACS lysing buffer. Store at 4°C for 24 hours and then acquire using flow cytometry. This step is to check effectiveness of CD25 depletion.